This invention relates to fluorescence detection, and more particularly, to a system capable of increased dynamic range and four-color detection.
Fluorescence readers are often used for re-sequencing or gene expression studies. In these systems, light such as that from a laser is directed onto a target, which may include molecules capable of fluorescing. Of course, the light could come from the process of chemi-luminescence as well. The emitted fluorescent light is then detected and analyzed. Oftentimes, multiple color dyes are utilized. For example, four colors may be used. The light is detected by florescence detection devices such as confocal scanning microscopes and imagers that utilize detection elements such as photomultiplier tubes (PMTs), avalanche photo-diodes (APDs), and charge-coupled devices (CCDs).
Different users, or the same user performing different experiments, may require different operating modes of the detection equipment. If a particular slide employs four color dyes, then four separate detectors can be used to detect each of the four colors. If, however, there are only two dyes on a slide, the experimenter may wish increased dynamic range in the output with respect to the two dyes. Heretofore, two separate instruments would be required in order both to detect four separate colors or to detect two colors with a higher dynamic range. Alternatively, multiple scans would have had to be used with, for example, a two-color/two-detector instrument. See, U.S. Pat. No. 6,078,390. The present invention is directed to a single versatile instrument that can operate selectively in these two modes.
In one aspect, the light detection system of the invention includes at least two light detection elements with each element responsive to a selected light color in a first mode. In a second mode, each element has a different sensitivity within a dynamic range and each is responsive to the same single color.
In another aspect, the light detection system includes four light detection elements with each element responsive to a selected color when operated in a first mode. In a second mode, the four light detectors are grouped into two pairs of detection elements with each element in a pair having a different sensitivity from the other element in the pair for increased dynamic range for detection of a color. When operating in the second mode, it is preferred that one element has a sensitivity near the element""s detection limit at the low end of a dynamic range and the other element has a sensitivity near the element""s maximum non-saturating signal limit at the high end of the dynamic range. It is also preferred that there be overlap in the elements"" sensitivities to cover a dynamic range continuously.
In preferred embodiments, the light detector includes photomultiplier tubes, charge-coupled devices and avalanche photo-diodes. When a photomultiplier tube or an avalanche photo-diode is used, the voltage is altered to adjust its sensitivity to either the low or high end of the dynamic range.
The system of the invention may include a moveable mirror/beam splitter arrangement to switch between the first and second modes or, alternatively, dichroic filters may serve as fractional beam splitters in another embodiment.
The instrument according to the invention can thus be used to detect multiple colors or to detect fewer colors with higher dynamic range. The instrument is entirely general and may be extended, for example, to 6 PMT""s for 6 color detection or for 3 color detection with extra dynamic range. The invention also includes 4 PMT""s, for example, for either 4 colors or 4 times more dynamic range for one color.